The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

Fetal bovine serum-a cell culture dilemma.

Ethical and possible reproducibility issues arise when using fetal bovine serum in cell culture media.

Fetal Bovine Serum (FBS): Past - Present - Future.

The supplementation of culture medium with fetal bovine serum (FBS, also referred to as "fetal calf serum") is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations has gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where regulatory aspects, the serum dilemma, alternatives to FBS, case-studies of serum-free in vitro applications, and the establishment of serum-free databases were discussed. The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with the above-mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically-defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.

Fetal Bovine Serum (FBS) - A pain in the dish?

The use of Fetal Bovine Serum in replacement alternative methods is associated with serious animal welfare concerns, as well as worrying reproducibility issues.

A framework program for the teaching of alternative methods (replacement, reduction, refinement) to animal experimentation.

Development of improved communication and education strategies is important to make alternatives to the use of animals, and the broad range of applications of the 3Rs concept better known and understood by different audiences. For this purpose, the Center for Alternatives to Animal Testing in Europe (CAAT-Europe) together with the Transatlantic Think Tank for Toxicology (t(4)) hosted a three-day workshop on "Teaching Alternative Methods to Animal Experimentation". A compilation of the recommendations by a group of international specialists in the field is summarized in this report. Initially, the workshop participants identified the different audience groups to be addressed and also the communication media that may be used. The main outcome of the workshop was a framework for a comprehensive educational program. The modular structure of the teaching program presented here allows adaptation to different audiences with their specific needs; different time schedules can be easily accommodated on this basis. The topics cover the 3Rs principle, basic research, toxicological applications, method development and validation, regulatory aspects, case studies and ethical aspects of 3Rs approaches. This expert consortium agreed to generating teaching materials covering all modules and providing them in an open access online repository.

Effects of the introduction of in vitro assays on the use of experimental animals in pharmacological research.

The introduction of in vitro assays in pharmacological research has led to a reduction in the number of experimental animals used. But what has been the degree of this reduction, and when did it really start? This report describes the events in a medium-sized pharmaceutical company. Analysis of data collected over the last 12 years shows a five-fold reduction in the number of experimental animals used per compound synthesised. Compounds from compound libraries (large collections of randomly-synthesised molecules) that are being assessed for potential bioactivity in 'high-throughput screening' were not included in this analysis. Over the years, the (average) degree of discomfort for the animals in the experiments did not vary much; with variation generally observed from 1.5 to 2.0 (on a scale from 1-6). There was a peak in the discomfort score of experimental mice in 1997, which could be explained by the initiation of arthritis models that were subsequently refined, resulting in a lower degree of suffering. It might be concluded that the introduction of in vitro assays has indeed brought about a significant reduction in the number of experimental animals required to select a good compound (i.e. one that could progress to the preclinical toxicology phase). However, this development appears to have been neutralised by the low survival rate of new chemical entities in clinical studies, leading to a lower number of compounds per annum that actually reach the market place. Put in this 'productivity perspective', the number of experimental animals required to select a marketable drug has not much changed in the last decade.

Proposal for the composition of animal experiments committees in the Netherlands.

The Dutch Act on Animal Experimentation (1996) requires that local animal experiments committees (AECs) review animal experiments and balance the scientific and societal benefits of the experiments against the suffering caused to the animals used. Each AEC is composed of at least seven members who provide a balance of expertise in animal experiments, alternatives to laboratory animal experiments, ethics, and animal welfare and protection. This study proposes selection criteria for individuals possessing each of the four AEC required areas of expertise. Criteria were established minding that, on the one hand, sufficient knowledge and expertise can be demonstrated whilst, on the other hand, a sufficient number of people would qualify to participate in the AECs. The results of this study may serve as a starting point for further discussion of selection criteria for members of AECs both in the Netherlands and in other countries where ethical review processes have been or are being implemented.

ESTIV questionnaire on the acquisition and use of primary human cells and tissue in toxicology.

The ability to use human cells and tissues in toxicology research and testing has the benefit that it obviates the need to undertake species extrapolation when assessing human hazard. However, obtaining and using human cells and tissues is logistically difficult, ethically complex and is a potential source of infections to those coming into contact with human cell material. The issue is also controversial, with the recent EU legislation draft on tissue engineering, and also due to some instances of human material being obtained and used without informed consent. There are also varying regulations and attitudes relating to the use of human cells and tissues throughout Member States of the EU, and there is a need for harmonisation. The European Society of Toxicology in Vitro (ESTIV) Executive Board and the European Network of Human Research Tissue Banks (ENRTB) have conducted a survey to ascertain the extent to which human cells and tissues are used by its members, how these are obtained, what local regulations are in force, how the material is used, and the advantages and disadvantages experienced by members in using such material, as opposed to cell lines. The results obtained have been compared with the results from a previous survey conducted in 2000. It is hoped that this information will help to facilitate the process of acquiring and using human cells and tissues in a safe and effective way to promote the use of non-animal approaches for investigating the mechanisms of toxicity, and for predicting the toxic hazard of substances.

Fourth meeting of the European Network of Research Tissue Banks - future strategy to increase collaborations in the supply of human tissue for biomedical research.

This report records the Fourth meeting of the European Network of Research Tissue Bank (Brussels, 18th March 2004) which was attended by Mel Read MEP. The existing membership of this informal group represents European Human Research Tissue Bankers, biomedical researchers seeking access to human tissue and allied groups including animal welfare representatives. This Fourth meeting provided a forum to update members on individual activity in this area. A particular focus of this meeting was to consider the status of this group and future affiliations to increase the profile and activity of this Network. This meeting addressed differences in legislative and ethical requirements governing the use of human tissue in biomedical research in the different countries represented. Future activity of the ENRTB, planned at this meeting, will target harmonisation of current differences which are currently barriers to increased access to human tissue for biomedical research. Through the harmonisation of procurement, processing and distribution of human tissue specimens the ENRTB will provide a mechanism to benefit human health through increased use of human tissue in pharmacotoxicological studies and the associated replacement of animal tests.

Reduction through education: the insight of a trainer.

One of the articles contained within European Council Directive 86/609/EEC states that "Persons who carry out experiments or take part in them, and persons who take care of animals used for experiments, including duties of a supervisory nature, shall have appropriate training". In effect, this article stipulates that only competent individuals are allowed to work with laboratory animals. At least three groups of individuals can be identified with different responsibilities toward experimental animals: animal technicians, scientists, and veterinarians/animal welfare officers. The responsibilities and duties of the individuals within each of these categories differ. This paper focuses on the training of scientists. The scientist designs, and often also performs, animal experiments. Therefore, scientists must be educated to develop an attitude of respect toward laboratory animals, and must be trained so that, if an experiment must be performed with animals, it is designed according to the highest possible scientific and ethical standards. In The Netherlands, the law stipulates that scientists intending to work with animals must have completed a course in laboratory animal science. This compulsory course started in 1986. The Department of Laboratory Animal Science at Utrecht University is responsible for the national coordination of this course. Participants must have an academic degree (at the level of MSc) in one of the biomedical sciences, such as biology, medicine or veterinary medicine. Although the course is an intensive 3-week, 120-hour long course, which covers both technical and ethical aspects of laboratory animal experimentation, it cannot provide full competence. It is designed to provide sufficient basic training and knowledge to enable students to design animal experiments, and to develop an attitude that will be conducive to the implementation of the Three Rs. However, full competence will always require further training that can only be acquired as a result of practical experience gained while working in the field of laboratory animal research. Evaluations subsequent to the course have revealed that more than 98% of the students regard the course as indispensable for all scientists working in a research area where animal experiments are performed. They agree that the course not only contributes to the quality of experiments and to the welfare of animals, but also to a decrease in the number of animals used in experiments.

Report and recommendations of the international workshop "Retrieval approaches for information on alternative methods to animal experiments".

In the member states of the EU and in the USA, scientists are obliged by animal welfare legislation not to conduct an animal experiment if another scientifically satisfactory method is reasonably and practicably available. To meet the regulatory obligation to use alternatives to animal experiments, scientists should consult literature and other relevant sources on alternatives prior to any experimental study on laboratory animals. It is the responsibility of the individual scientist to select the most appropriate database to obtain information on alternatives, which have been defined as methods that refine, reduce or replace animal experiments (the 3 Rs concept of Russell and Burch (1959)). Specialised information services provide support to scientists searching for publications on alternative methods. On occasion of a workshop in Berlin in November 2003, representatives of animal welfare information centres discussed currently available information sources on alternative methods, index terms for alternative methods, and search strategies based on index terms for alternative methods. ZEBET presented an investigation on the current status of indexing systems on alternative methods in established literature databases. The project analysed how the results of a search for publications on alternatives was influenced by the indexing procedure. The results of the study were exemplified by a typical search result. The results of the study indicated that the current indexing systems do not provide the required information, since not all of the relevant information is indexed under "alternative methods". The workshop participants developed recommendations for ad hoc working groups and research projects, e.g. development of suitable search strategies on alternative methods for scientists.

The European Resource Centre for Alternatives in Higher Education.

The European Resource Centre for Alternatives in Higher Education (EURCA: http://www.eurca.org) is an exciting new project, which aims to enable teachers using animals in teaching to be more creative and innovative in their approach to teaching and learning, to foster high-quality training for science students, and to significantly reduce the number of animals used, often unnecessarily, in teaching. This will be achieved by: a) establishing a resource centre--a collection of mainly electronic alternatives, and taking this to relevant scientific meetings in Europe, where it would function as a drop-in advice centre for teachers; b) creating a network of academic teachers who actively use alternatives, to take responsibility for disseminating information about alternatives to other teachers in the European Union, to participate in the activity outlined above, and to share experiences and good practice; c) setting up an Internet website with an expansive, information-rich database (peer-reviews, demos, peer-evaluations, peer-recommendations, links to users, etc.) on selected "tried and tested" alternatives; and d) encouraging and promoting the findings of evaluative studies on the effectiveness of alternatives in higher education teaching and learning.

The use of fetal bovine serum: ethical or scientific problem?

Fetal bovine serum (FBS) is a common component of animal cell culture media. It is harvested from bovine fetuses taken from pregnant cows during slaughter. FBS is commonly harvested by means of a cardiac puncture without any form of anaesthesia. Fetuses are probably exposed to pain and/or discomfort, so the current practice of fetal blood harvesting is inhumane. Apart from moral concerns, several scientific and technical problems exist with regard to the use of FBS in cell culture. Efforts should be made to reduce the use of FBS or, preferably, to replace it with synthetic alternatives.